1. Field of the Invention
The present invention relates to an imaging flow cytometer. More specifically, the invention relates to an imaging flow cytometer in which, for instance, to detect a particular substance in a cell or a microorganism, a suspension (called a sample liquid) of cells or microorganisms that have been reacted with a fluorescence-dyed or fluorescence-marked monoclonal antibody or DNA probe, for instance, is introduced into a thin glass tube, and a fluorescence image of a cell, a microorganism is taken by a flow scheme and analyzed.
2. Description of the Related Art
In recent years, molecular-biological analyses and tests have been widely conducted for diagnoses of cancer, hereditary diseases, etc. and analyses of cell dynamism. For example, to detect a particular substance (DNA, a propagative antigen, HBsAg, a cancer gene product, or the like) included in a cell, blood cell, or the like, or to conduct a test for abnormality in the number of chromosomes, cells are processed using a specific reaction reagent that is a fluorescence-marked monoclonal antibody, chromosome-specific DNA probe, or the like and fluorescence images of such cells are examined with a fluorescence microscope.
To evaluate the safety of medical supplies, food additives, etc., micronucleus tests have been conducted to check existence of fragments (micronuclei) that are produced by abnormal chromosomes. In the micronucleus test, micronuclei remaining in blood cells are subjected to fluorescence dying and the existence of micronuclei is checked using a fluorescence microscope, or the like.
Further, to test the degree of voraciousness of white blood cells, beads marked by a fluorescent substance have been caused to be gormandized by white blood cells and the number of beads is counted using fluorescent images.
Conventionally, since the above measurements have been performed by using a fluorescence microscope, much time and labor was needed if a large number of cells or blood cells were involved.
In a measurement using a fluorescence microscope, a small depth of field is not a serious problem in the case where cells develops on a slide glass. However, in a measurement on a sample in which a cell-floating liquid is sealed in a slide glass, a small depth of field needs to be compensated such that a measurer observes cells at every point along the optical axis while varying the focal point of a microscope.
Recently, it was attempted to automatize the above analyses and tests by combining a fluorescence microscope and an image processing device. However, it took long time for such an apparatus to analyze a number of cells. Although measurements using the conventional flow cytometer (FCM) were also attempted, this method could not provide information on the locality of a part emitting fluorescence. In addition, the measurement accuracy was affected by background fluorescence (unspecific fluorescence) from parts other than a part where a specific substance to be detected existed. Therefore, this method has not been put to practical use yet.
To solve the above problems, an apparatus called a fluorescence image capturing type imaging flow cytometer (IFCM) has been put to practical use. In this apparatus, a suspension of cells or microorganisms that have been reacted with a specific fluorescence-dyed or fluorescence-marked monoclonal antibody or DNA probe is introduced into a thin glass tube, and fluorescence images of the cells or microorganisms are taken and subjected to analysis.
However, where this apparatus is applied to the above measurements, due to a small depth of field, it is difficult for this apparatus to measure, with high accuracy, light emitting points that are distributed three-dimensionally in a cell.
Therefore, various attempts are made to overcome the small depth of field. One example is a method of increasing the depth of field (i.e., obtaining a larger depth of field) optically. In this method, NA of an image-forming optics is set as small as possible; for example, a depth of field of more than 12 .mu.m is obtained with NA of 0.25. However, this method cannot secure three-dimensional resolution because, for instance, two light emitting points arranged parallel with the optical axis are superimposed and observed as a single light emitting point. That is, light emitting points cannot be counted correctly, meaning lowered measurement accuracy.
There is a paper relating to this invention: "Study of Fluorescence in situ Hybridization for Detection of Chromosome Aberration," Human Cell 2(4), pp. 436-438, 1989. The abstract of this paper states "The authors applied fluorescence in situ hybridization (FISH) technique for the detection of chromosome aberration in interphase nuclei using the probe specific to alphoid repeats on chromosome 11 and X. Chromosome 11 specific probe showed two major spots in lymphocyte nuclei, while X specific probe showed single spot in male and double spots in female respectively. On the other hand three spots were detected in most of the nuclei from HeLa cells with 11 and X specific probes. We concluded that FISH with the use of chromosome specific probe may become a useful and reliable tool for the detection of chromosome aberration in interphase nuclei."
Further, a disclosure to the effect that a plurality of particle images of a single cell are taken at different time points with different focal positions is found in Cell Analysis, Vol. 1, pp. 306-313, 1982.